Rice Genetics IV - IRRI books - International Rice Research Institute

Antisense and cosuppression RNA. Expression of antisense RNA in transgenicplants has been extensively used to inhibit gene expression (see van den Elzen et al1989, Finnegan and McElroy 1994). In animal systems, antisense has been reportedto operate by either preventing the processing or transport of RNA in the nucleus orhybridizing to the target mRNA in the cytoplasm and preventing translation (Green etal 1988). Huntley and Hall (1996) transformed rice with several PDR genes designedto express (1) an artificial DI RNA derived from RNA-2, (2) a sense tRNA-like structurecorresponding to the 3’ end of RNA-2, (3) an antisense sequence correspondingto the intercistronic region of BMV RNA-3, and (4) RNA encoding the viral capsidprotein. When inoculated with virion RNA, protoplasts (obtained from transgenicplants or callus lines) showed up to 95% reduction in accumulation of progeny viralRNAs. The addition of 20 times the normal level of inoculum was required to overcomethe induced resistance. The observed interference appeared to be mediatedthrough viral RNAs rather than protein products.There is increasing evidence that the antisense RNA in plants is inducing a posttranscriptionalgene-silencing mechanism (PTGS), the same mechanism that is inducedby cosuppression RNA (Baulcombe 1996). PTGS (see Finnegan and McElroy1994, Flavell 1994) was first proposed as a mechanism for induced virus resistance inplants by Lindbo and Dougherty (1992) from their work on tobacco transformed withpotyvirus coat protein genes. They observed that the plants showing high levels ofresistance had low levels of coat protein mRNA in the cytoplasm yet their transgeneswere being highly transcribed in the nucleus. Their conclusions were that very highlevels of transcription of the transgene (usually associated with high transgene copynumber) induce a mechanism in the plant that specifically degrades the mRNA of thattransgene and, as the transgene mRNA has the same sequence as part of the viralgenome, the virus genome is also degraded by the induced mechanism (see Baulcombe1996, Stam et al 1997).Posttranscriptional gene silencing. PTGS is frequently observed in transgenicplants in which active transcription of a transgene results in silencing of its homologousgenes or resistance to the virus from which the transgene sequence is derived.With conventional sense and antisense constructs, gene silencing or virus resistanceusually occurs in only a small proportion of a transgenic population. Recent studieshave shown that these plants often contain transgene integration as an inverted repeat,in which the direction of transcription is toward the center of the repeats (Stam et al1997, Wang and Waterhouse 2000). Recently, we have discovered that an invertedrepeat transgene, designed to produce RNA with internal self-complementarity, wasexceptionally effective at inducing PTGS and conferring virus resistance in plantscompared with conventional sense and antisense transgenes (Waterhouse et al 1998,1999, Wang and Waterhouse 2000). We have also found that co-expression of a senseand an antisense strand of a viral sequence in plants can confer complete immunity tothe virus (Waterhouse et al 1998). These findings have demonstrated that doublestrandedRNA is an important inducer of PTGS in plants and offers a novel strategyfor engineering virus resistance in plants.Engineering for virus resistance in rice 409